不同诱导时间下大鼠脂肪间充质干细胞成骨的差异

发布时间：2018-05-12 05:25

本文选题：种子细胞 + 脂肪间充质干细胞　；参考：《山西医科大学》2011年硕士论文

【摘要】：目的:观察诱导时机的不同对大鼠脂肪间充质干细胞成骨分化的影响。方法:脂肪间充质干细胞(ADSCs)来源于SD大鼠,雌雄不分,体重在150-200g(由山西医科大学实验动物中心提供)。取其腹股沟处脂肪垫,分离、纯化、培养,传至第三代,将细胞分为诱导组和非诱导组。诱导组又分为,在细胞接种同时(A组),50%-60%融合时(B组),85%-95%融合时(C组)分别加入同一浓度的由地塞米松、维生素C和β-甘油磷酸钠组成的等量诱导剂诱导。非诱导组的A、B、C组由普通培养基培养不加入诱导剂。在诱导后的7,14,21,28 d进行钙钴染色、茜素红染色、碱性磷酸酶活性的检测来判断各组细胞的成骨能力,并且定期在倒置显微镜下观察细胞形态变化及生长状况。结果: 1.相同初始接种密度,诱导组细胞生长速度略慢于同期非诱导组。 2.经钙钴染色后,诱导组大部分染色的细胞呈阳性反应,细胞胞浆中可见浅棕色至棕黑色的细胞颗粒。相同初始接种密度,同期比较,干预时融合度越高阳性细胞越多,以细胞达85%-95%融合时诱导组出现结节数量最多、体积最大;非诱导组未见片状强阳性细胞及明显结节形成。 3.茜素红染色后,诱导组从第7d开始可见少量散在红色结节,到28d呈现大片红色致密结节。同期相比,以诱导组细胞达85%-95%融合时形成结节早,且结节数量多、体积大,非诱导组未见明显阳性结节形成。 4.细细胞内碱性磷酸酶活性检测表明:诱导组与同期非诱导组相比细胞内碱性磷酸酶活性明显提高(P0.05),表现细胞成骨分化特点。诱导组组内比较,第7天A组与B组无明显差别(P0.05),与C组比较有明显差别(P0.01),C组高;第14与21天A、B、C组间比较均有差异(P0.01),C组B组A组;第28天A组与B、C组比较有差异(P0.05),A组低,B组与C组比较无明显差异(P0.05)。非诱导组内各组比较无明显差异(P0.05)。结论: 1.ADSCs在体外一定条件下经过诱导剂诱导后,可向成骨细胞方向分化。 2.初始接种密度相同的情况下,细胞间相互接触程度越高,成骨能力越强。
[Abstract]:Aim: to observe the effect of different induction time on osteogenic differentiation of adipose mesenchymal stem cells in rats. Methods: adipose mesenchymal stem cells (ADSCs) were derived from SD rats, male and female, weight 150-200g (provided by Experimental Animal Center, Shanxi Medical University). The cells were separated, purified, cultured and transferred to the third generation. The cells were divided into induction group and non-induced group. The induction group was also divided into two groups: group A and group B were induced by dexamethasone, vitamin C and sodium 尾 -glycerophosphate at the same concentration. No inducer was added to the normal culture medium in AZB group (non-induction group). Calcium cobalt staining, alizarin red staining and alkaline phosphatase activity were used to determine the osteogenic ability of the cells at 28 days after induction, and the morphological changes and growth status of the cells were observed under inverted microscope. Results: 1. At the same initial inoculation density, the cell growth rate of the induction group was slightly slower than that of the non-induction group. 2. After calcium and cobalt staining, most of the cells in the induction group showed positive reaction, and light brown and black cell granules were found in the cytoplasm of the cells. At the same initial inoculation density, at the same time, the higher the fusion degree, the more positive cells were in the intervention, and the number and volume of the nodules in the induction group were the largest when the cells reached 85% -95%, but no flake strong positive cells and obvious nodule formation were found in the non-induction group. 3. After alizarin red staining, a small number of red nodules were seen in the induction group from the 7th day, and a large number of red dense nodules appeared on the 28th day. At the same time, 85% to 95% of the cells in the induction group formed the nodules early, and the number and volume of the nodules were large. No positive nodules were found in the non-induced group. 4. The detection of alkaline phosphatase activity in fine cells showed that the activity of alkaline phosphatase in the induction group was significantly higher than that in the non-induction group at the same time, showing the characteristics of osteogenic differentiation. There was no significant difference between group A and group B on the 7th day, but there was a significant difference between group C and group C, and there was significant difference between group A and group B on the 14th and 21st days. On the 28th day, there was no significant difference between group A and group B (P 0.05). There was no significant difference between the groups in the non-induction group (P 0. 05). Conclusion: 1.ADSCs can differentiate into osteoblasts after induced by inducer under certain conditions in vitro. 2. Under the same initial inoculation density, the higher the intercellular contact, the stronger the osteogenic ability.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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